中华皮肤科杂志 ›› 2021, Vol. 54 ›› Issue (3): 189-195.doi: 10.35541/cjd.20200895

• 论著 • 上一篇    下一篇

白念珠菌菌丝调控小鼠巨噬细胞自噬关键分子微管相关蛋白1轻链3的实验研究

林泽杭    段志敏    徐松    陈旭    李岷   

  1. 中国医学科学院、北京协和医学院皮肤病研究所  江苏省皮肤病与性病分子生物学重点实验室，南京  210042
  • 收稿日期:2020-09-09 修回日期:2021-01-08 发布日期:2021-03-02
  • 通讯作者: 陈旭；李岷 E-mail:chenx@pumcderm.cams.cn; limin@pumcderm.cams.cn
  • 基金资助:
    国家自然科学基金（81773338）；江苏省自然科学基金（BK20190144）；南京市国家级临床医学中心培育计划项目（2019060001）；中国医学科学院医学与健康科技创新工程项目（2017-I2M-1-017）

Regulatory effect of Candida albicans hyphae on the key autophagy-related molecule microtubule-associated protein 1 light chain 3 in murine bone marrow-derived macrophages

Lin Zehang, Duan Zhimin, Xu Song, Chen Xu, Li Min   

  1. Institute of Dermatology, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China
  • Received:2020-09-09 Revised:2021-01-08 Published:2021-03-02
  • Contact: Chen Xu; Li Min E-mail:chenx@pumcderm.cams.cn; limin@pumcderm.cams.cn
  • Supported by:
    National Natural Science Foundation of China （81773338）; Natural Science Foundation of Jiangsu Province （BK20190144）; The Nanjing Incubation Program for National Clinical Research Center （2019060001）; CAMS Innovation Fund for Medical Sciences （2017-I2M-1-017）

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摘要： 【摘要】 目的 探讨白念珠菌菌丝对小鼠骨髓来源巨噬细胞（BMDM）自噬流的影响。方法 白念珠菌菌丝分别体外诱导BMDM细胞0.5、4、12 h，以不加菌丝处理的0 h组作为对照，Western印迹法检测各时间点自噬关键蛋白微管相关蛋白1轻链3（LC3）-Ⅰ向LC3-Ⅱ的转换及磷酸化雷帕霉素机制性靶蛋白（p-mTOR）的表达。白念珠菌菌丝分别联合4种溶酶体阻断剂，包括半胱氨酸蛋白酶抑制剂E-64d + 胃蛋白酶抑制剂pepstatin、巴弗洛霉素-A1（BAF-A1）、氯化铵及氯喹，体外诱导小鼠BMDM细胞4、12 h，观察白念珠菌菌丝对BMDM细胞基础自噬流的影响。统计分析采用非配对t检验、析因设计的方差分析及LSD-t检验。结果 白念珠菌菌丝体外处理小鼠BMDM 0.5、4和12 h后，与0 h组（0.983 ± 0.030）相比，LC3-Ⅰ向LC3-Ⅱ转换均增加（1.254 ± 0.118、1.629 ± 0.391、1.598 ± 0.379），差异有统计学意义（t值分别为3.875、2.856、2.804，均P < 0.05），但各组p-mTOR的蛋白表达无明显差异。白念珠菌菌丝联合E-64d + pepstatin体外处理BMDM细胞4和12 h后，LC3-Ⅱ的蓄积水平较E-64d + pepstatin单独处理组明显增高，差异均有统计学意义（t值分别3.691、6.648，均P < 0.05）。与相应溶酶体阻断剂组相比，白念珠菌菌丝联合BAF-A1、氯化铵或氯喹4和12 h后，LC3-Ⅱ的蓄积水平均显著升高（均P < 0.05）。结论 白念珠菌菌丝体外诱导可增加小鼠BMDM细胞基础自噬流中LC3-Ⅰ向LC3-Ⅱ的转换。

关键词: 白色念珠菌, 菌丝, 巨噬细胞, 自噬, 微管相关蛋白质类

Abstract: 【Abstract】 Objective To evaluate the effect of Candida albicans （C. albicans） hyphae on autophagic flux in murine bone marrow-derived macrophages （BMDM）. Methods BMDM were in vitro stimulated with C. albicans hyphae for 0.5, 4 and 12 hours, and the 0-hour group treated without hyphae served as a control. Western blot analysis was performed to detect the conversion of microtubule-associated protein 1 light chain 3 （LC3）-Ⅰto LC3-Ⅱ, and determine the expression of phosphorylated mechanistic target of rapamycin （p-mTOR） at each time point. Some BMDM were divided into several groups: control group receiving no treatment, hyphae group treated with C. albicans hyphae, lysosomal inhibitor groups treated with different lysosomal inhibitors, including E-64d （a cysteine proteinase inhibitor） + pepstatin （a pepsin inhibitor）, bafilomycin-A1 （BAF-A1）, ammonium chloride and chloroquine, and hyphae combined with lysosomal inhibitor groups treated with lysosomal inhibitors immediately followed by C. albicans hyphae. After 4- or 12-hour treatment, the effect of C. albicans hyphae on basal autophagic flux in murine BMDM was evaluated. Statistical analysis was carried out by using unpaired t test, factorial design analysis of variance and least significant difference-t test. Results After 0.5-, 4- and 12-hour in vitro treatment with C. albicans hyphae, the conversion of LC3-Ⅰto LC3-Ⅱ significantly increased in murine BMDM （1.254 ± 0.118, 1.629 ± 0.391, 1.598 ± 0.379, respectively） compared with the 0-hour group （0.983 ± 0.030; t = 3.875, 2.856, 2.804, respectively, all P < 0.05）, while there was no significant difference in the protein expression of p-mTOR among the 0-, 0.5-, 4- and 12-hour groups. After 4- and 12-hour in vitro treatment with C. albicans hyphae combined with lysosomal inhibitors E-64d and pepstatin, the accumulation level of LC3-Ⅱ significantly increased in BMDM compared with those treated with E-64d and pepstatin alone （t = 3.691, 6.648, respectively, both P < 0.05）. Compared with the corresponding lysosomal inhibitor groups, the accumulation level of LC3-Ⅱsignificantly increased in BMDM treated with C. albicans hyphae combined with BAF-A1, ammonium chloride or chloroquine for 4 and 12 hours（all P < 0.05）. Conclusion In vitro treatment with C. albicans hyphae can increase the conversion of LC3-Ⅰto LC3-Ⅱ in the basal autophagic flux in murine BMDM.

Key words: Candida albicans, Hyphae, Macrophages, Autophagy, Microtubule-associated proteins